The pharmacology and molecular pharmacology of two groups of gastrointestinal (GI)peptides were investigated. VIP-related peptides [VIP-related peptides [VIP, PACAP] have widespread effects in the GI tract and central nervous system. The actions of VIP-PACAP-related peptides are mediated by three receptors (VPAC1, VPAC2, PACAP-R). Studies in this project are aimed at understanding the molecular basis of action of these different receptors and at developing selective metabolically stable ligands that function as agonists or antagonists. To define the pharmacology of VIP receptors, the VPAC1 and VPAC2 receptors have been cloned and stably transfected in CHO and PANC1 cells. The VIP pharmacophore is being determined for each VPAC-R subtype by alanine and D-amino acid screening, to attempt to identify selective ligands. Last year results for the VPAC1 were reported and this year we have completed the definition of the human and rat VPAC2 pharmacophore. Amino acids in position 3,6,7,10,12,22 and 23 of VIP were essential for high affinity whereas side chains of amino acids in 2,8,9,16,19,20,21,24, and 25 were not essential. Comparison with VPAC1 show side chains in position 7,10,11,22 were more important for high affinity for VPAC2. There were significant species differences between rat and human VPAC2. This study provides a basis for development of simplified VIP analogues and selective ligands for each VPAC subtype. Bombesin-related peptides (gastrin-releasing peptide [GRP], neuromedin B) interact with two distinct receptors (GRP-R, NMB-R) to mediate a number of effects in the GI tract and central nervous sytem (CNS). The aims of this project are to understand the pharmacology, molecular pharmacology, and cell biology of these receptors. Our results demonstrate the critical amino acids in the 3rd extracellular domain (EC-3) are responsible for GRP selectivity. Particularly important is the substitution of the Ile for Phe185 and Ile for Ala198 in the GRPR compared to the NMBR. Site-directed mutagenesis studies showed this selectivity was primarily mediated by hydrogen bonding and pi-cation interactions between these ligands and the receptor. Our results suggest an interaction between the aromatic ring of Phe185 of the GRPR with GRP is the most important for GRP selectivity. Similar studies are now underway with the NMBR.